Pathogens responsible for many of the common human infectious diseases such as urinary tract infection (UTI), gastroenteritis, pneumonia, and wound infections have proven to be highly adept in acquiring mechanisms of antimicrobial resistance. Widespread injudicious practice of empiric antibiotic usage by healthcare providers and infiltration of antibiotics in the food chain have accelerated selection and dissemination of resistant pathogens. As a consequence, clinicians have fewer treatment options, particularly in the most needy patients. An example of the problem was the rapid emergence of trimethoprim- sulfamethoxazole (SXT) resistant E. coli, which accounts for 85-90% of the UTIs in the community setting. Prior to the 1990s, beta-lactams such as ampicillin (AMP) were the standard antimicrobial regimen for acute uncomplicated UTIs, but was replaced with SXT when E. coli resistance against beta-lactams surpassed 25%. With increasing use, however, SXT resistance increased substantially and quinolones such as ciprofloxacin (CIP) became the antibiotic of choice. Not surprisingly, quinolone-resistant uropathogens are on the rise. In hospitals where MDR pathogens are of even greater problem, the quinolone-resistance rate for uropathogenic E. coli has now exceeded 50% in some settings. The goal of this Phase II NIAID Advanced Technology SBIR application is to develop and validate RAST (rapid antimicrobial susceptibility testing), an integrated diagnostic compact system to enable clinicians to direct point-of-care (POC), evidence-based selection of antibiotics for treatment of acute bacterial infections. RAST addresses the major limitations of standard phenotypic AST platforms (e.g., bioMerieux Vitek, BD Phoenix) by providing rapid (90 minutes vs. 2 days) and decentralized (POC vs. laboratory-based) testing. RAST complements our ongoing NIAID Cooperative Agreement, An Integrated Diagnostic Biochip for Point of Care Pathogen Identification (U01 AI082457), for rapid molecular diagnosis urinary tract infections (UTI) using electrochemical biosensors integrated with microfluidics. Since Phase I, we have accomplished several critical milestones: (1) development and clinical validation of a 3.5 hour bench-top RAST protocol showing 94% accuracy; (2) compatibility of RAST with clinical urine samples without need for initial bacterial isolation; (3) feasibility of on-chip electrokinetic bacterial concentration and assay enhancement; (4) on-chip bacterial culture using microchannels; (5) integrated microfluidic cartridge for pathogen identification; and (6) preliminary feasibility of cartridge-based RAST. In the current Phase II project, we propose three Specific Aims: Specific Aim 1. Optimization and validation of electrokinetic (EK) processing modules for volume reduction and in situ assay enhancement. The goal of Aim 1 is to develop an EK volume reduction module for enriching the sample 100-fold within 10 min and to develop an in situ EK enhancement technique for improving the detection sensitivity of the electrochemical assay by 10-fold. Specific Aim 2. Development of the RAST cartridge for rapid phenotypic antimicrobial susceptibility testing. The goal of Aim 2 is to develop the process flow and fabrication process for the RAST cartridge and reader/manifold system, including sample loading, EK volume reduction, on-chip sample culturing in selective media containing different antibiotics of interest, and phenotypic AST by quantitative measurement of bacterial 16S rRNA. Specific Aim 3. Clinical translation of RAST cartridge in urine. The goal of Aim 3 is to perform analytical validation of RAST cartridge and reader/manifold system and a clinical feasibility study using 30 unknown clinical samples from patients suspected to have UTI. The development and validation of RAST will adhere to the recommended standards of Quality Management Standard for Medical Devices (ISO 13485) and federal regulations for fully automated short-term incubation cycle antimicrobial susceptibility system (21 CFR 866.1645)(see Commercialization Plan D.1.3). Successful accomplishment of our milestones in this Phase II application will be followed by FDA 510(k) submission to demonstrate the system is substantially equivalent to a predicate device. A separate Milestones and Timeline section is included at the end of the Research Strategy.